1. Field of the Invention
The present invention relates generally to the field of fat metabolism and weight control. More specifically, the present invention relates to the role of the ACC2 isoform of acetyl-CoA carboxylase in regulating fatty acid accumulation and oxidation.
2. Description of the Related Art
Acetyl-CoA carboxylase (ACC), a biotin-containing enzyme, catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate metabolite that plays a pivotal role in the regulation of fatty acid metabolism. It has been found that malonyl-CoA is a negative regulator of carnitine palmitoyltransferase I (CPTI, a component of the fatty-acid shuttle system that is involved in the mitochondrial oxidation of long-chain fatty acids. This finding provides an important link between two opposed pathways-fatty-acid synthesis and fatty-acid oxidation. Thus, it is possible to interrelate fatty acid metabolism with carbohydrate metabolism through the shared intermediate acetyl-CoA, the product of pyruvate dehydrogenase. Consequently, the roles of malonyl-CoA in energy metabolism in lipogenic (liver and adipose) and non-lipogenic (heart and muscle) tissues has become the focus of many studies.
In prokaryotes, acetyl-CoA carboxylase is composed of three distinct proteins-the biotin carboxyl carrier protein, the biotin carboxylase, and the transcarboxylase. In eukaryotes, however, these activities are contained within a single multifunctional protein that is encoded by a single gene.
In animals, including humans, there are two isoforms of acetyl-CoA carboxylase expressed in most cells, ACC1 (Mrxcx9c265,000) and ACC2 (Mrxcx9c280,000), which are encoded by two separate genes and display distinct tissue distribution. Both ACC1 and ACC2 produce malonyl-CoA, which is the donor of the xe2x80x9cC2-unitsxe2x80x9d for fatty acid synthesis and the regulator of the carnitine palmitoyl-CoA shuttle system that is involved in the mitochondrial oxidation of long-chain fatty acids. Hence, acetyl-CoA carboxylase links fatty acid synthesis and fatty acid oxidation and relates them with glucose utilization and energy production, because acetyl-CoA, the substrate of the carboxylases, is the product of pyruvate dehydrogenase. This observation, together with the finding that ACC1 is highly expressed in lipogenic tissues such as liver and adipose and that ACC2 is predominantly expressed in heart and skeletal muscle, opened up a new vista in comparative studies of energy metabolism in lipogenic and fatty acid-oxidizing tissues.
Diet, especially a fat-free one, induces the synthesis of ACC""s and increases their activities. Starvation or diabetes mellitus represses the expression of the Acc genes and decreases the activities of the enzymes. Earlier studies addressed the overall activities of the carboxylases with specific differentiation between ACC1 and ACC2. Studies on animal carboxylases showed that these enzymes are under long-term control at the transcriptional and translational levels and short-term regulation by phosphorylation/dephosphorylation of targeted Ser residues and by allosteric modifications induced by citrate or palmitoyl CoA.
Several kinases have been found to phosphorylate both carboxylases and to reduce their activities. In response to dietary glucose, insulin activates the carboxylases through their dephosphorylation. Starvation and/or stress lead to increased glycogen and epinephrine levels that inactivate the carboxylases through phosphorylation. Experiments with rats undergoing exercises showed that their malonyl-CoA and ACC activities in skeletal muscle decrease as a function of exercise intensity thereby favoring fatty acid oxidation. These changes are associated with an increase in AMP-kinase activity. The AMP-activated protein kinase (AMPK) is activated by a high level of AMP concurrent with a low level of ATP through mechanism involving allosteric regulation and phosphorylation by protein kinase (AMP kinase) in a cascade that is activated by exercise and cellular stressors that deplete ATP. Through these mechanisms, when metabolic fuel is low and ATP is needed, both ACC activities are turned off by phosphorylation, resulting in low malonyl-CoA levels that lead to increase synthesis of ATP through increased fatty acid oxidation and decreased consumption of ATP for fatty acid synthesis.
Recently, it was reported that the cDNA-derived amino acid sequences of human ACC1 and ACC2 share 80% identity and that the most significant difference between them is in the N-terminal sequence of ACC2. The first 218 amino acids in the N-terminus of ACC2 represent a unique peptide that includes, in part, 114 of the extra 137 amino acid residues found in this isoform. Polyclonal antibodies raised against the unique ACC2 N-terminal peptide reacted specifically with ACC2 proteins derived from human, rat, and mouse tissues. These findings made it possible to establish the subcellular localization of ACC1 and ACC2 and to later demonstrate that ACC2 is associated with the mitochondria and that the hydrophobic N-terminus of the ACC2 protein plays an important role in directing ACC2 to the mitochondria. ACC1, on the other hand, is localized to the cytosol.
Although these findings and the distinct tissue distribution of ACC1 and ACC2 suggest that ACC2 is involved in the regulation of fatty acid oxidation and that ACC1 is involved in fatty acid synthesis primarily in lipogenic tissues, they do not provide direct evidence that the products of the genes ACC1 and ACC2 have distinct roles.
These distinctions between the two ACC isoforms could not have been predicted prior to the generation of the Acc2 knockout mouse described herein. Moreover, malonyl-CoA, the product of the ACC1 and ACC2, seems to be present in the liver and possibly in other tissues in two separate pools that do not mix and play distinct roles in the physiology and metabolism of the tissues. Malonyl-CoA, the product of ACC1, is involved in fatty acid synthesis as the donor of xe2x80x9cC2-carbons.xe2x80x9d On the other hand, malonyl-CoA, the product of ACC2, is involved in the regulation of the carnitine palomitoyl CoA shuttle system, hence in the oxidation of fatty acids. This functional distinction between the roles of the products of ACC1 and ACC2 based on the results obtained with the Acc2 mice was not expected nor could it have been predicted prior to this study.
Moreover, the current study demonstrates that ACC2, through its product malonyl-CoA, is potentially an important target for the regulation of obesity. Inhibition of ACC2 would reduce the production of malonyl-CoA, leading to continual fatty acid oxidation and energy production. This continual oxidation of fatty acid would be achieved at the expense of freshly synthesized fatty acids an d triglycerides and of body fat accumulated in the adipose and other fatty tissues leading to reduced body fat.
The prior art is deficient in an understanding of the separate roles ACC1 and ACC2 have in the fatty acid metabolic pathways. The prior art is also deficient in assigning the differential roles of the malonyl-CoA generated by ACC1 versus that generated by ACC2 in regulating fatty acid metabolism. Also, the prior art is deficient in transgenic knockout mice generated to lack ACC2 and methods of using these transgenic mice. The present invention fulfills this long-standing need and desire in the art.
Malonyl-CoA (Maxe2x80x94CoA), generated by acetyl-CoA carboxylases ACC1 and ACC2, is the key metabolite in the regulation of fatty acid (FA) metabolism. Acc1xe2x88x92/xe2x88x92 mutant mice were embryonically lethal, possibly due to a lack of xe2x80x9cC2-unitsxe2x80x9d for the synthesis of fatty acid needed for biomembrane synthesis. Acc2xe2x88x92/xe2x88x92 mutant mice bred normally and had normal life spans. Acc2xe2x88x92/xe2x88x92 mice fed normal diets did not accumulate fat in their livers as did the wild-type mice and overnight fasting resulted in a 5-fold increase in ketone bodies production, indicating higher fatty acid oxidation. ACC1 and fatty acid synthase activities and malonyl-CoA contents of the livers of the Acc2xe2x88x92/xe2x88x92 and Acc2+/+ mice were the same, indicating that fatty acid synthesis is unperturbed, yet the malonyl-CoA was not available for the inhibition of the mitochondrial fatty acid shuttle system, hence fatty acid oxidation was relatively high. This result was not predicted earlier to this finding, and it is very important in distinguishing the roles of the malonyl-CoA generated by ACC1 versus that generated by ACC2 in regulating fatty acid metabolism.
Absence of ACC2 resulted in 30- and 10-fold lower malonyl-CoA contents of muscles and heart, respectively. Fatty acid oxidation in the Acc2xe2x88x92/xe2x88x92 soleus muscles was 30% higher than that of ACC2+/+ mice. Addition of insulin did not affect fatty acid oxidation in the Acc2xe2x88x92/xe2x88x92 soleus muscle, but, as expected, it did reduce fatty acid oxidation by 50% in the wild-type soleus muscle compared to that of the mutant. This is a very important observation since it demonstrates for the first time the role of ACC2 in insulin action and regulation of fatty acid oxidation in diabetes. Isoproterenol, an analog of glucagon, had little effect on fatty acid oxidation in the muscle of the Acc2xe2x88x92/xe2x88x92 mice but caused a 50% increase in fatty acid oxidation in the soleus muscle. Again, this result highlights the important role of ACC2 in regulating fatty acid oxidation and its potential as a target for the regulation of obesity. The higher fatty acid oxidation in the mutant mice resulted in a 50% reduction of fat storage in the adipose tissue compared to that of the wild-type mice. These results are valuable to an understanding and control of fatty acid metabolism and energy homeostasis in normal, diabetic, and obese animals, including humans.
In one embodiment of the instant invention, a method of promoting weight loss and/or fat oxidation in an individual is provided. This method may comprise the administration of an inhibitor of acetyl-CoA carboxylase 2 (ACC2) to the individual. The same method may be used for weight loss as well.
In yet another embodiment of the instant invention, a method is provided for promoting fatty acid oxidation to treat conditions such as obesity and diabetes comprising the administration of an inhibitor of acetyl-CoA carboxylase 2 (ACC2) to an individual having such conditions.
In another embodiment of the instant invention, a method of decreasing blood sugar by administering an inhibitor of acetyl-CoA carboxylase 2 (ACC2) to an individual is provided. This method may be used to treat an individual with diabetes.
In another embodiment of the present invention, there is provided a transgenic mouse having a mutation in an endogenous gene for the ACC2 isoform of acetyl-CoA carboxylase that inactivates the protein. The ACC2 gene may be mutated by deleting one or more exons of the gene, which may be replaced by heterologous DNA sequences such as an HPRT expression cassette. In a preferred embodiment, an exon encoding a biotin-binding motif of ACC2 is replaced with an HPRT expression cassette. Unexpectedly to those in the field, these mice exhibit a phenotype consisting of a reduction in malonyl-CoA levels in skeletal muscle and heart, unrestricted fat oxidation, and reduced fat accumulation in the liver and fat storage cells. The transgenic mice consume more food than wild-type mice but remain lean.
In yet another embodiment of the present invention, there is provided a method of screening for an inhibitor of ACC2 isoform activity consisting of the step of administering potential inhibitors to wild-type mice and screening for mice that exhibit the same phenotype of the Acc2xe2x88x92/xe2x88x92 transgenic mice.
In yet another embodiment of the present invention, there is provided an ACC2 inhibitor identified by the above method. This inhibitor may be incorporated into a pharmaceutical composition to be administered to individuals for purposes of augmenting fatty acid oxidation and inhibiting fat accumulation to promote weight loss or maintenance.
The present invention has further potential for the treatment of diabetic animals, including humans, in that it may help insulin-administered type I and type II diabetics from gaining weight. Furthermore, increased fatty acid oxidation would affect carbohydrate metabolism by increasing glycolysis, and reducing gluconeogenesis and glycogen synthesis and accumulation of fatty acid oxidation independent of insulin. Thus it helps diabetics to burn fat and lose weight.
In a further embodiment of the instant invention, a method is described for obtaining a purified preparation of ACC1 protein totally free of the ACC2 isoform by purifying ACC1 from the Acc2xe2x88x92/xe2x88x92 transgenic mice.
In another embodiment of the instant invention, a method is provided for obtaining improved antibodies against ACC2 by generating the antibodies in the Acc2xe2x88x92/xe2x88x92 transgenic mice.
In yet another embodiment of the instant invention, cell lines derived from the Acc2xe2x88x92/xe2x88x92 transgenic mice are provided. Cell lines derived from muscle, heart, adipose cells, and liver cells are expected to be especially useful in bioassays and drug targeting studies. Brain cell lines including those of the hypothalamus would be useful in studying the neuropeptides involved in regulating feeding behavior and appetite and fat and carbohydrate metabolism.
In yet another embodiment of the present invention, a method of screening for agonists and antagonists of ACC2 is provided. This method comprises the steps of administering candidate compounds to Acc2xe2x88x92/xe2x88x92 cell lines and to cell lines derived from wild-type mice followed by experiments to detect alterations in cellular activity. A compound that specifically acts on ACC2 will alter cellular activity, fat and carbohydrate metabolism in wild-type cells but have no effect on Acc2xe2x88x92/xe2x88x92 cells. Cellular activities that may be monitored include mRNA expression, protein expression, protein secretion, and catalytically active proteins (enzymes) involved in fatty acid and lipid and carbohydrate metabolism.
The absence of Ser 1201 in ACC2 represents an important difference between ACC1 and ACC2 regulation and can b e advantageous in designing and/or generating differential inhibitor(s) [drug(s)] for ACC1 and ACC2. Other and further aspects, features, and advantages of the present invention, including the unique hydrophobic amino-terminal of ACC2, will be advantageous in designing and/or generating differential inhibitor(s) [drug(s)] for ACC1 and ACC2. Also, the differential reactions of ACC2 to anti-ACC1 antibodies would be important in designing and generating differential inhibitors for ACC1 and ACC2. Moreover, further aspects will be apparent from the following description of the embodiments of the invention given for the purpose of disclosure.